Abscisic acid d6

Phytohormones were quantified as described previously (Vadassery et al., 2012 (link); Meena et al., 2019 (link)). Seedlings were sampled at 2 dpi and 14 dpi. The samples were harvested, frozen immediately in liquid N₂, lyophilized, and ground to a fine powder. Weighed, powdered samples (25 mg) were extracted using 1.5 ml of methanol containing internal standards of 60 ng d₆-jasmonic acid (HPC Standards GmbH, Cunnersdorf, Germany), 60 ng salicylic acid-d₄ (Santa Cruz Biotechnology), 60 ng abscisic acid-d₆ (Toronto Research Chemicals), and 12 ng d₆-jasmonic acid-isoleucine conjugate (HPC Standards GmbH). A triple-quadruple LC-MS/MS system was used for phytohormone quantification (SCIEX 6500).

Ethanol (≥ 99.9%, LiChrosolv^®), tetrahydrofuran (THF, ≥ 99.9%, LiChroSolv^®), n-hexane (SupraSolv^®), dichloromethane (< 99.8%, SupraSolv^®), chlorophyll a and b (analytical standards) and norflurazone (Pestanal^®, analytical standard) were obtained from Merck KGaA (Darmstadt, Germany). Tert-butyl methyl ether (≥ 99.9%, Rotisolv^®), 2-propanol (≥ 99.9%, Rotisolv^®), ammonium acetate (≥ 98%) and acetic acid (100%, Supra Quality) were purchased from Carl Roth GmbH (Karlsruhe, Germany). MEthanol (Chemsolute^®) and acetonitrile (Chemsolute^®) were purchased from Th. Geyer GmbH & Co. KG (Renningen, Germany). Carotenoid standards were obtained from CaroteNature GmbH (Münsingen, Switzerland) and flavonol glycosides from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). Abscisic acid (ABA) standard was purchased from Sigma Aldrich Chemie GmbH (Taufkirchen, Germany) and (+)-abscisic acid-d6, ≥ 98%) from Toronto Research Chemicals (North York, Canada). All solvents were of LC-MS quality and the water was of ultra-pure quality.

For metabolite analysis, roots of two plants from each glass jar were pooled and ground to a fine powder in liquid nitrogen. For the analysis of A/S, 35 mg of lyophilized powdered root was extracted in 1.5 mL of methanol in an ultrasonic bath at 10% power for 3 h (Bandelin Sonorex Digital 10P, Berlin, Germany) followed by centrifugation for 10 min at 12,500 rpm (Hermle Z 216 MK, Wehingen, Germany). The supernatant was collected and then filtered with 0.22 μm syringe filters. For phytohormonal analysis, ~ 10 mg of the lyophilized root powder was dissolved in 1 mL of methanol containing the mix of the following chemicals as internal standards: D6-JA (40 ng, HPC Standards GmbH, Germany), D4-SA (40 ng, Santa Cruz Biotechnology, USA), D6-abscisic acid (40 ng, Toronto Research Chemicals, Toronto, Canada), D6-JA-isoleucine conjugate (8 ng, HPC Standards GmbH, Germany), and D5-indole-3-acetic acid (OlChemIm s.r.o., Olomouc, Czech Republic). After mixing for 30 min at room temperature, the samples were centrifuged at 4 °C and 14,000 rpm for 20 min. The supernatant was used for LC–MS/MS analysis. Sample preparation for chiral analysis of alkannin and shikonin are described in Supplementary Methods.